JPH09140373A - New fungal strain belonging to exserohilum monoceras and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new fungal strain belonging to Exserohilum monoceras exhibiting a specific esterase zymogram pattern which is not observed in well- known fungal strains, having high weeding effect and spore-producing ability and useful as a component, etc., for microbial weed controlling agents. SOLUTION: This fungal strain is a new fungal strain of Exserohilum monoceras JTB-012 (FERM BP-5271), JTB-013 (FERM BP-5272), JTB-799 (FERM BP-5273), JTB-803 (FERM BP-5274) or JTB-808 (FERM BP-5275) exhibiting esterase zymogram pattern different from fungal strains belonging to well-known Exserohilum monoceras, having high weeding effect and spore-producing ability and useful as a component, etc., for microbial weed controlling agents. The fungal strain is obtained by selecting fungal strain having high weeding effect and high spore producing ability from the fungal strains having pathogenicity against barnyard grass.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an excellohirum
The present invention relates to a novel strain belonging to Exserohilum monoceras, a weed control agent and a weed control method using the same.

[0002]

2. Description of the Related Art The use of pesticides is indispensable in agricultural land for stable food production. However, since a large amount of a chemically synthesized pesticide containing a chemical substance that does not exist in nature is sprayed on an agricultural land for a long period of time, various harmful effects have appeared. The most important problems are the direct impact of pesticides on humans and animals and the destruction of ecosystems. In recent years, the specificity of chemical pesticides against weeds, pests, and pathogens has dramatically improved, so the impact on humans and animals has been reduced, but sufficient attention should be paid to the opportunity to come into contact with high-concentration chemicals when spraying pesticides. It is still necessary. Furthermore, chemical pesticides do not necessarily have high selectivity for target weeds, pests, and pathogens, and thus often disturb the ecosystem.

In order to solve the above problems, there has been a demand for the development of environmentally friendly pesticides that have no influence on non-target species. One of them is a herbicide that uses weed pathogenic microorganisms. Weed pathogenic microorganisms are ubiquitous in weeds, and microbial herbicides that use them are less likely to cause harm to small animals such as humans, seafood, and insects, and are unlikely to cause medicinal damage to plants. It is known. In addition, since the microbial herbicide has high specificity for the target weed, it is considered to have high selectivity and low possibility of disturbing the ecosystem. Currently, commercialized microbial herbicides include:
Devine, Colletotrichum gloeo, a herbicide for Strangler vines using Phytophthora palmivora
Northern join using sporioides f.sp. aeschynomene
Colleg, a herbicide for tvetch (a closely related species of Kusanem)
o, and Colletotrichum gloeosporioides f.sp. mal
round-leaved mallow with vae
BioMal herbicide.

In the paddy field, Cochliobolus lunatus (Anamorph: Curvularia lunata) [Weed Research (198
7), 27, 43-47, JP-A-5-284963), or Ust
ilago trichophora (WO93 / 05656), Drechslera monoce
ras (a synonym for Exserohilum monoceras) [JP-A-3-219883
JP, JP 4-226905 JP, JP 4-360678 JP, JP 4-370090 JP, JP 6-277042 JP, JP 6-329513 JP, JP 6-247822 JP ] The control agent using is known.

[0005] However, Cochliobolus lunatus requires a dew point time of 18 hours or more in order to exert a sufficient effect, and Ustilago trichophora requires 4 to 5 weeks after application of the drug until a sufficient effect appears, and Drechslera mon
Each oceras has a problem of low spore production. Therefore, none of the above-mentioned control agents using microorganisms has been put into practical use.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a microorganism exhibiting a sufficient herbicidal effect and having a high spore producing ability.

[0007]

Means for Solving the Problems The present inventors selected a strain having a high herbicidal effect from strains having a pathogenicity to Nobie, and found that in addition to having a high herbicidal effect, a spore-producing ability was also high. A high group of strains was found. Furthermore, when their intracellular esterase zymogram patterns were examined, it was found that all the strains in the group showed a similar staining pattern, and that the staining pattern was not similar to any staining pattern of known strains. . Based on the above knowledge, the present inventors have completed the following inventions.

That is, the present invention is a strain belonging to Excellohirum monoceras showing the esterase zymogram pattern shown in FIG. Further, the present invention is a weed control agent containing the above-mentioned strain as an active ingredient. Furthermore, the present invention is a weed control method using the above-described weed control agent.

Hereinafter, the present invention will be described in detail. The strain of the present invention was isolated from diseased Nobie and its mycological properties are as follows. It is an aerobic bacterium, and the colony is dark gray or black on potato sucrose agar medium. Also, white or gray aerial hyphae may be seen. Conidia are dark-colored, multivesicular, with 2-8 inner and outer septa, spindle-shaped, with the widest central part, narrowing toward both sides. In addition, a navel (hilum) projects at the base end. The size of conidia is 40-150 × 10
It is within 25 μm.

The above results are shown in "GRAMINICOL by A. SIVANESAN.
OUS SPECIES OF BIPOLARIS, CURVULARIA, DRECHSLERA,
EXSEROHILUM AND THEIR TELEO MORPHS '' (Mycological P
apers, No.158, p-261, Nov.1987), p201-237 showed that the above strains were exserohilum monoceras due to the fact that the shape of the colony and the morphology of conidia were the same. ) Was identified.

Incidentally, the genus name of the genus Excellohirum is A.
It follows the classification of SIVANESAN. Also, LUTTRELL
(Revue de Mycologie Volume 41, 271-279, 1977) and AL
CORN (Mycotaxon Volume 8 411-414, 1978) also supports this taxonomy. On the other hand, Ellis (Dematioceous Hyphomyc
etes, CMI, Kew, 608, 1971) described the genus Exerochirum and the genus Bipolaris as Drechs.
Lera) is included only in the genus Drexurera. However, in recent years, there are no researchers other than Ellis who do not recognize the genus Exerohilum and the genus Bipolaris, so it is considered appropriate to adopt the genus Exerohilum. Also, the complete generation of the genus Excellohirum is a genus of Setosphaeria
Since it is known to be Haeria), microorganisms classified as the genus Setosphaeria are also included in the technical scope of the present invention.

The strain of the present invention is specifically shown in JTB-
012, JTB-013, JTB-799, JTB-8
03, JTB-808. JTB-
012 is FERM BP-5271 (deposit date October 27, 1995)
Sun), JTB-013 is FERM BP-5272 (deposit date October 27, 1995), JTB-799 is FERM BP-5273 (deposit date October 27, 1995), JTB-803 is FERM BP-.
5274 (deposit date October 27, 1995), JTB-808
Has been deposited at the Institute of Biotechnology, Institute of Biotechnology, with a deposit number of FERM BP-5275 (deposit date October 27, 1995).

As shown in FIG. 2, these strains are IFO9, which is a known strain belonging to Exerohirum monoceras.
800, IMI125854, IMI125855, ATCC24641, ATCC5834
It shows an esterase zymogram pattern different from 6. It is not necessary to use a special method for culturing this strain, and it can be cultivated by the same method as that for a known strain belonging to Exerohirum monoceras. As the medium, either a synthetic medium or a natural medium can be used as long as it appropriately contains an assimilable carbon source, a nitrogen source, an inorganic substance and a necessary growth promoting substance. As a specific medium,
Examples thereof include oatmeal sucrose agar medium, oatmeal agar medium, potato sucrose agar medium, V-8 juice agar medium, Tuapeck-Dox agar medium and the like.
In culturing, the temperature is 15 to 30 ° C., preferably 20.
It is desirable to maintain the pH at -25 ° C and the pH at 3-9, preferably 5-8. When cultured for about 7 to 14 days under the above conditions, a sufficient amount of spores are formed on the surface of the medium.

The weed control agent of the present invention is prepared by using the above-mentioned spores as an active ingredient and adding a surfactant and the like thereto. Although the spore concentration can be arbitrarily determined within the range where the herbicidal effect is exhibited, it is 10 ² to 10 ⁶ spores / ml, preferably 10 ³
It is suitable to set the concentration to -10 ⁵ spores / ml. When the weed control agent of the present invention is applied to an actual field, it is preferable to apply it so that the amount of spores is 10 ⁹ to ¹⁰ 10 per 10 ares.

The weed-controlling agent of the present invention is mainly used for Nobie, but is not limited thereto.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017]

【Example】

[Example 1] Novie controlling effect test After collecting diseased Nobie from all over Japan, the excised lesions were incubated according to a conventional method in a humid chamber at 25 ° C to form conidia. The conidia were taken with a handle and placed on a potato-sucrose agar medium to separate monospores. Inoculate each strain on oatmeal sucrose agar and
Culture was performed for 4 days to form conidia. After this,
The spores were suspended in a 0.02% Tween20 aqueous solution, and then the suspension was prepared so as to have 10 ³ , 10 ⁴ , and 10 ⁵ spores per 5 ml.

On the other hand, after growing Nobye (20 individuals per pot) in a pot of 1 / 10,000 are until the leaf stage of 1.5 leaves, it was flooded to a depth of about 5 cm. The above spore suspension was added dropwise to each pot in an amount of 5 ml and allowed to stand in a greenhouse for 3 weeks, and the control effect was investigated. The Herbicidal effect of Nobie was evaluated as shown below. Control effect = [1- (number of surviving individuals / 20)] × 100 Table 1. Strain and Novier control effect ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Strain name Control effect ─────── ──────────────── 10 ³ 10 ⁴ 10 ⁵ ───────────────────────────── ─────── JTB-012 38 95 100 JTB-013 35 70 95 JTB-799 47 100 100 JTB-803 35 100 100 100 JTB-808 32 100 100 100 IMI-125855 0 2 25 No treatment 0 ──── ───────────────────────────────

[Example 2] Conspore-producing ability test Each strain was inoculated into oatmeal sucrose agar medium,
The cells were cultivated at 25 ° C. for 14 days. Then, the conidia on the medium were recovered by suspending them in 0.1% Tween20. The conidia production is shown in Table 2. Table 2. Strain and conidia production capacity ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━ Strain name Conidia production / cm ² ─────────────────────────────────── JTB-012 4.4 × 10 ⁵ JTB-0134 .5 × 10 ⁵ JTB-799 7.5 × 10 ⁵ JTB-803 8.6 × 10 ⁵ JTB-808 6.6 × 10 ⁵ IFO-9800 <2.4 × 10 ³ IMI-125854 <2.4 × 10 ³ IMI-125855 4.0 × 10 ⁴ ATCC-24646 <2.4 × 10 ³ ATCC-58346 <2.4 × 10 ³ ──────────────────── ──────────────── JTB-012, JTB-013, JTB-799, J
The conidial production of the strain group consisting of TB-803 and JTB-808 was 4.4 × 10 ⁵ spores / cm ² or more. On the other hand, among the known strains, the strain having the highest conidial spore-producing ability was IMI-125855, and the conidial production amount was 4.0 × 10 ⁴ spores / cm ² . The conidial production of all other known strains was below the detection limit. From this, the strains produced 1 conidiospores compared with known strains.
It became clear that it was more than 0 times higher.

Example 3 Analysis of Esterase Zymogram JTB-012, JTB-013, JTB-799, J
TB-803, JTB-808, IFO-9800, I
MI-125854, IMI-125855, ATCC
-24641, 10 strains of ATCC-58346 were used as samples. Preparation of a crude enzyme solution of each strain is described in JP-A-6-32.
According to the method of 9513 gazette.

Each strain was placed in a potato sucrose medium at 25 ° C.
The cells were cultivated in the dark for 7-10 days to form a cell mat. The obtained bacterial cell mat was washed with distilled water several times, and then -80
After freezing at ℃, it was freeze-dried. The cells were disrupted in 50 mM Tris-hydrochloric acid buffer (pH 7.4), filtered through a filter paper, the filtrate was centrifuged at 10,000 rpm, and the supernatant was used as a sample. The protein concentration in the sample was quantified by the method of Lowry. Approximately 50 μg per sample using acrylamide gel (concentrated gel: 4.5%, separation gel 10%) in a large slab gel electrophoresis layer
Was electrophoresed at 30 mA for 2 hours.

After electrophoresis, esterase activity staining was performed. 40 mg of α-in 4 ml of 50% aqueous acetone solution
After dissolving naphthyl acetic acid, 200 mg of fast viole
t B salt and 200 ml of 50 mM Tris-hydrochloric acid buffer were added to prepare a staining solution. The gel was dipped in this staining solution and gently shaken to stain for 30 minutes. Then, the gel was washed with distilled water and the mobility of each band was measured.

As a result, as shown in FIGS. 1 and 2, J
TB-012, JTB-013, JTB-799, JT
Since the esterase zymogram patterns of B-803 and JTB-808 were all the same, JTB-01
2, JTB-013, JTB-799, JTB-80
3, JTB-808 is clearly the same strain group. In addition, this strain group is a known strain, IFO-98.
00, IMI-125854, IMI-125855,
ATCC-24641, ATCC-58346 and Drechs rela monoceras v shown in Japanese Patent Laid-Open No. 6-329513.
It was revealed that this strain group is a novel strain different from the known strains because it is different from the esterase zymogram pattern of ar. microsporus.

[Formulation Example] Formulation Example 1 (liquid formulation) Conidiospores (2 × 10 ⁹ pieces) of Exerohilum monoceras JTB-013 strain and Tween 80 (4 g) were added to 20 L of sterile water.
And mixed to prepare a liquid preparation. Formulation example 2 (wettable powder) 10 ⁷ conidia (JTB-803 strain) were suspended per 1 ml of a mixed solution of maltose 9%, clay 1% and water 90%. After this was air dried, the dried product was mixed and pulverized to prepare a wettable powder.

Formulation Example 3 (wettable powder) 1% of a mixed solution of lactose 9%, zeolite 1% and water 90%
10 ⁷ conidia (JTB-012 strain) were suspended per ml. After this was air dried, the dried product was mixed and pulverized to prepare a wettable powder.

Formulation Example 4 (Wettable Powder) 10 ⁷ conidia (JTB-808 strain) were suspended per 1 g of a mixed solution of diatomaceous earth 15%, kaolin 77% and polyoxyethylene alkylphenyl ether 8%. After this was air dried, the dried product was mixed and pulverized to prepare a wettable powder. Formulation Example 5 (wettable powder) 33% diatomaceous earth, 0.33 carboxymethyl cellulose
%, Water 66.67% Mixture Conidia (J
TB-799 strain 10 ⁷ cells were suspended. After this was air dried, the dried product was mixed and pulverized to prepare a wettable powder.

Formulation Example 6 (powder) Hydroxypropyl-β-cyclodextrin 14%,
10 ⁷ conidia (JTB-012 strain) were mixed per 1 g of a mixture of 12% white carbon and 74% clay. After drying, the powder was uniformly ground to prepare a powder.

Formulation Example 7 (Granule) 10 ⁷ conidia (JTB-808 strain) per 1 g of a mixture of 15% β-cyclodextrin, 2% starch, 18% bentonite, 36% calcium carbonate and 29% water. After kneading and kneading, the granules were prepared by granulating with a granulator and drying.

Formulation Example 8 (Emulsion) Polyoxyethylene nonylphenyl ether Ammonium phosphate 18%, polyoxyethylene nonylphenyl ether 6%, triethyl phosphate 29%, tributyl phosphate 47% per 1 g of a mixture of conidia (JTB) -799
10 ⁷ strains were added and suspended uniformly to prepare an emulsion.

Formulation Example 9 (oil formulation) Conidia (JTB-803) in 1 ml of a mixed solution of spindle oil 95%, castor oil 4% and silicone oil 1%.
An oil solution was prepared by suspending 10 ⁷ strains.

Formulation Example 10 (dry flowable agent) 10 ⁷ conidia (JTB-013 strain) were suspended in 1 ml of a composition of sodium alkylbenzenesulfonate 12% and polyethylene glycol ether 88% to prepare a dry flowable agent. did. Formulation Example 11 (Capsule) 10 ⁷ conidiospores (JTB-803 strain) were suspended in 1 ml of a mixed solution of sodium alginate 0.7%, kaolin 5%, glycerin 15%, and water 79.3%, to give 0.1%. A capsule-like product was obtained by dropping into a 2 molar calcium acetate solution. After chopping this, it was sieved and air-dried to prepare a capsule.

FORMULATION EXAMPLE 12 (Capsule) Sodium alginate 0.7%, diatomaceous earth 5%, glycerin 15%, water 79.3% In a mixture of 1 ml, conidia (J
(TB-013 strain) 10 ⁷ cells were suspended and added dropwise to a 0.2 molar calcium chloride solution to obtain a capsule-shaped product. After chopping this, it was sieved and air-dried to prepare a capsule.

[0033]

INDUSTRIAL APPLICABILITY The present invention provides a novel strain belonging to Exerohirum monoceras. This strain has a higher herbicidal effect and spore-producing ability than the conventional strains belonging to Exerohilum monoceras, and has suitable properties as a component of a microbial herbicide.

[Brief description of the drawings]

FIG. 1 is a diagram showing an esterase zymogram of the novel strain of the present invention.

FIG. 2 is a diagram showing esterase zymograms of a novel strain and a known strain of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tadaharu Hieda 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa Prefecture 6-2 Japan Tobacco Inc. Plant Development Laboratory Yokohama Center (72) Inventor Masatoshi Gohara, Aoba-ku, Yokohama, Kanagawa Prefecture 6-2 Umegaoka Japan Tobacco Inc. Plant Development Laboratory Yokohama Center

Claims (6)

[Claims] 1. A strain belonging to Exerohilum monoceras showing the esterase zymogram pattern shown in FIG. 2. Excellohilm monoceras JTB-0
12. The strain according to claim 1, which is 12, Excellohilum monoceras JTB-013, Excellorhilm monoceras JTB-799, Excellorhilm monoceras JTB-803, or Excellorhilm monoceras JTB-808. 3. A weed control agent, which comprises the strain according to claim 1 or 2 as an active ingredient. 4. The weed-controlling agent according to claim 3, wherein the weeds are Nobie. 5. A weed control method using the weed control agent according to claim 3. 6. The method for controlling weeds according to claim 5, wherein the weeds are Nobie.